The antigenically closely related viruses of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family transmit in nature in a cycle between ticks and various mammal species and cause human disease of varying severity with up to 30% mortality. Most of these viruses are Select Agents based on their high lethality and their potential for human infection by the oral or aerosol route. Currently, a vaccine produced by formalin inactivation of TBEV is available in Europe, but is not licensed in the US. The existing vaccine does not afford lifetime protection; booster immunizations are required to sustain high levels of immunity against TBEV. A live attenuated TBEV vaccine is expected to induce a higher level and more durable immunity than that induced by an inactivated virus vaccine. We developed a new approach to TBEV immunoprophylaxis, which involved construction of chimeric virus that contains the genes for structural proteins of a Langat virus (LGT, a member of the TBEV complex) or TBEV with the rest of the genome derived from mosquito-borne dengue-4 virus (DEN4). The resulting LGT/DEN4 chimera was (1) highly attenuated in mice, i.e., it exhibited greatly reduced neurovirulence and lacked neuroinvasiveness in SCID mice; (2) highly attenuated in rhesus monkeys; (3) immunogenic and efficacious in mice and monkeys; and (4) non-infectious for mosquitoes. As a result, a Phase I clinical testing of LGT/DEN4 vaccine in healthy adult volunteers was recently initiated at the Johns Hopkins School of Hygiene and Public Health Center for Immunization Research. To improve immunogenicity of our vaccine candidates against virulent TBEV viruses, two chimeras were generated by replacing the structural prM and E protein genes of DEN4 with the corresponding genes of a Far Eastern TBEV in the presence (TBEV/DEN4-del30) or absence (TBEV/DEN4) of a 30 nucleotide deletion (del30) in the 3? noncoding region of the chimeric genome. The addition of the del30 mutation to TBEV/DEN4 greatly attenuated the chimeric virus for SCID mice and rhesus monkeys and induced a higher level of antibody against the TBEV than did LGT/DEN4. The TBEV/DEN4-del30 appears to be a promising vaccine candidate. However, prior to use of the TBEV/DEN4-del30 as a live virus vaccine candidate in the clinical trials in humans, it will be necessary to examine its neurovirulence in monkeys. We have extended our flavivirus chimeric vaccine strategy to include West Nile virus (WN) and closely related St. Louis encephalitis virus (SLE). During the 1999-2004 outbreaks of WN in the USA, there were 16,637 WN illnesses reported that included 654 deaths. WN illness is considered to be an emerging disease in the USA and presents a significant public health threat since a human vaccine is not available. Goals of our project are to construct and evaluate WN/DEN4 and SLE/DEN4 chimeric viruses in humans as possible vaccines against disease caused by WN or SLE. We constructed a viable WN/DEN4 chimera in which the structural prM and E protein genes of WN were substituted for the corresponding genes of DEN4 that differed in amino acid sequence by 61.5% for prM and 55.9% for E. In order to generate a further attenuated derivative of WN/DEN4, we introduced a 30-nucleotide deletion in the 3' non-coding region of genome. Both chimeric WN/DEN4 and WN/DEN4-del30 viruses have been evaluated for safety, infectivity, and immunogenicity in mice, geese, horses, and rhesus monkeys and for infectivity in mosquitoes. Chimeras: (1) exhibited more than 100,000,000-fold restriction of replication in mouse brain compared to wild-type WN when inoculated IC; (2) failed to induce fatal encephalitis in adult Swiss or SCID mice inoculated IP; (3) were highly attenuated in geese, horses, and monkeys; (4) were immunogenic in mice, horses, and nonhuman primates; (5) provided complete protection of mice and monkeys against WN challenge, and (6) were restricted in their ability to replicate in mosquitoes. Clinical trials in humans are underway with the WN/DEN4-del30 vaccine.